Isolation of sterols from fats and oils



Patented June 29, .1943

' ISOLATION OF STEROLS FROM FATS AND OILS Lester Yoder, Ames, Iowa,assignor to Iowa State College Research Foundation, Ames,

corporation of Iowa Iowa, at

No Drawing Application June 15, 1940, Serial No. 340341 7 Claims.(Cl.260397.2)

This invention relates to the isolation of certain sterols from complexmixtures of fats and oils, and to their subsequent purification. Morespecifically it relates to the separation of sterols containing onereactive double bond from the fatty and oily mixtures in which theyoccur.

An example of a sterol containing one reactive double bond ischolestrol, which is obtained from animal sources such as wool grease,nerve tissue fat, etc. Isa-cholesterol, a mixture of lanosterol andagnosterol, is obtained from wool grease and resembles cholesterol inmany respects. Phytosterol, another example, is found in vegetable oilssuch as pine oi-l, tall oil, corn oil etc.

' The processes used at present to separate these sterols from thematerials with which'they are mixed in nature are expensive anddiflcult. It is a purpose of this invention to provide an economical,practical, commercial method for their extraction. Fatty or oilymixtures containing the above sterols will hereinafter be termedsubstrates.

It is desirable, before the process of this invention is applied to asubstrate, that the sterol content of the substrate be in the form offree alcohols. It is also desirable, although not essential, that alarge bulk of the saponiflable fraction of the substrate be removed. Inmany substrates, such as nerve tissue fat and tall oil, the sterolsappear naturallyin this form. In

others, such as wool fat, a substantial fraction of the sterols appearin the form of esters and a preliminary treatment is necessary torecover these .fractions.

.Where esters of the desired sterols are present, their conversion toalcohols can be brought about by the saponiflcation of the substratewith a suitable basic ion, such as sodium or potassium hydroxide. Thesaponified portion is preferably separated from the unsaponifledfraction at this point and the latter retained for treatment inaccordance with the process of this invention, or, at the option of theoperator, the basic ion may be neutralized by the addition of acid ionand the entire mixture subjected to the process herein set forth.

The starting point of my process may be a substrate containing one ormore sterols which have one reactive double bond and which are in theform of free alcohols. This substrate is dissolved in some substantiallyanhydrous solvent. Because of the solubility in them of additionproducts formed in a later step, alcohol solvents are not suitable. Norshould a solvent be used which reacts readily with th acid used in alater what less than is necessary to dissolve the substrate to a largeexcess over that amount. However, a large excess of solvent tends tolower the yield of sterols.

To the dissolved or partially dissolved substrate I add a molar excessof a strong, monobasic, anhydrous acid which does not contain oxygen.The acid should b in a form in which it is soluble or miscible in thesolvent. Because of the ease with which they may be dissolved, I prefergaseous acids, such as the hydrogen halides, for this reaction. Hydrogenchloride is especially suitable because of its cheapness,

solubility, ease of handling and absence of sidereactions.

The optimum temperature for this reaction for a particular combinationof sterol, solvent and acid may easily be determined by one skilled inthe art. I prefer temperatures between about 5 C. and about 20 C.- Atlower temperatures there is a gradually increasing tendency toward theprecipitation of other products from the solution of substrate, while athigher temperatures the solubility of the acid in the solvent isdecreased, side reactions of the acid with the solvent and othermaterials present are increased, and the yield of sterols is reduced.

The result of the above treatment with acid is the precipitation fromthe solution of sterolacid addition'products, which, at the temperaturessuggested, are relatively insoluble in the solvents. This crystalliineprecipitate can be easily separated from the solution by filtrationorotherwise.

Addition of a molar excess of an alkaline ion to the precipitatedsterol-acid addition product. removes the acid therefrom, leaving thefree sterol. While it is not necessary, I find it convenient to add thealkaline ion in aqueous solution since the insolubility of the sterolsin water makes the elimination of the salt solution formed very simple.

If two sterols, each containing one reactive double bond, are present inthe same substrate,

such as, for example, cholesterol and iso-cholesterol in wool grease, Iuse the following procedure to separate them:

I dissolve the mixed precipitate of cholesterolacid addition product andiso-cholesterol-acid addition product obtained above in one of mysolvents, which may or may not contain some of the acid used toprecipitate the addition products. Solution is obtained by heating thesolvent to a sufficiently high temperature. large crystals ofcholesterol-acid addition productcrystallize and may be collectedseparately before the micro-crystals of iso-cholesterol-acid additionproduct crystallize. Then I neutralize each fraction with alkaline ionas shown above.

The following examples are illustrative of my invention:

Example 1 One hundred parts of a non-saponiflable fraction 'ofcommercialwool grease, containing 25 parts of free cholesterol, were dissolved in500 parts of ethylene chloride and the solution cooled to 10 C. A molarexcess of gaseous hydrogen chloride was passed into the solution, whichthickened to a paste. After one-halt hour at the low temperature, thecrystalline precipitate was filtered out and redissolved, at atemperature of 70 0., in ethylene chloride containing dissolved hydrogenchloride gas. deposited large crystals of cholesterol-hydrochloridewhich were filtered out. After a period of further cooling and standing,small crystals" of iso-cholesterol-hydrochlorlde were removed from thefiltrate. Each portion was treated with a 4 per cent solution of sodiumhydroxide and washed, producing 22 parts of cholesterol and 13 parts ofiso-cholesterol in the form of alcohols.

Example I! tralizing solution, 0.9 part of cholesterol remained.

- Example 111 I Forty parts of nonsaponiflable oil containing 1 parts ofphytosterol was dissolved in 130 parts of acetone, cooled to 15 C. andhydrogen bromide gas added in excess. The phytosterol hydrobromideseparated rapidly and, after a period, of

I time, was filtered off and treatedwith excess 5 per cent solution ofsoda ash and washed, producing 8 parts of phytosterol. I

1. In a process for the 'separation,'from oily or fatty substrates, ofsterols having one reactive double bond in the hydrophenanthrenenucleus, the steps of subjecting wool grease to saponifleation,separating the saponiflable and unsaponifiable portions thereof,dissolving the unsaponiflable portion in 'a tat-dissolving non-alcoholicn cooling, the- On cooling, the solution (In cooling the solutiondeposited 1 part of large crystals of cholesterol.-hydrochloride. Afterremoval 01' the aqueous alkaline neu asaaooa solvent, treating thesolution with a strong, monobasic, anhydrous acid which contains nooxygen. to form a precipitate of acid addition products of the-sterols,separating the precipitate from the solution, and treating theprecipitate with an alkaline ion.

2. A process for the separation of sterols having one reactive doublebond in thehydrophenanthrene nucleus from wool grease comprising,subjecting the wool grease to saponiflcation, separating thesaponiflable from the unsaponifiable portions thereof, dissolving theunsaponiflable portion in a fat dissolving non-alcoholic solvent,treating the solution with a hydrogen halideat a temperature between C.and 20 C. to form a precipitate of hydrohalic acid addition products ofthe sterols, separating the precipitate, and treating the precipitatewith an alkaline ion.

3. In a process for the separation, from nonsaponifiable fraction ofoils or fatty substrates, of sterols having one reactive double bond inthe hydrophenanthrene nucleus, the steps of .dissolving such substratein a fat-dissolving nonalcoholic solvent and dissolving a hydrogenhalide in such solution to precipitate the sterol acid addition producttherefrom.

4. In a, process for the separation from the nonsaponifiable fraction ofoily or fatty substrates; of sterols having one reactive double bond inthe ing such substrate ina fat-dissolving non-alcoholic solvent, andtreating such solution with a strong monobasic anhydrous acid whichcontains no oxygen to precipitate the sterol acid addition producttherefrom.--

5. In a process for the separation from the nonsaponifiable fraction ofoily or fatty substrates, of sterols having one reactive double bond inthe hydrophenanthrene I nucleus, the steps of dissolving such substratein a fat-dissolving nonalcoholic solvent, and treating such solutionswith a hydrogen halide at a temperature between about 5 C. and about 20C. to precipitate from the solution the sterol acid addition product.

saponifiable fraction of oily or fatty substrates, of

sterols having one reactive double bond inthe hydrophenanthrene nucleus,the steps of dissolving such substrate in a fat-dissolving nonalcoholicsolvent, and dissolving hydrogen chloride in such solution at atemperature between about 5 C. and about 20 C. to precipitate the Isterol acid addition product therefrom. I

7. A process for the separation of cholesterol in a fat-dissolvingnon-alcoholic solvent, and

treating such solution with a hydrogen halide to I hydrophenanthrenenucleus, the steps of dissolv- 6. In a process for the separation fromthe nonfrom the non-saponifiable fraction of oily or fatty substrates,comprising dissolving such substrate CERTIFICATE or CORRECTION. PatentNo. 2,322,9 6. June '29, 1915.

LESTER YYODER. r.

It is hereby certified that error appears in the printed specificationor the above numbered patent requiring correction as follows: Page 1,first column, line 8, for "cholestrol" read --cho1esterol--; page 2,second column, 1ine 20, claim '5, for the Word "oils" read-wily"; andthat the said Letters Patent should be read with this correction thereinthat the same may conform to the record of the case inthe Patent Office.

Signed and sealed this 10th day of August, A. D. 1914.}.

Henry Van Arsdale, (Seal) Acting Commissioner of Patents.

